Caratterizzazione Elettrochimica di Wildtype e Mutanti di CbA5H con Voltammetria Ciclica su Film Proteico

[FeFe]-Hydrogenases are constantly studied as biological catalyst for reversible hydrogen production. The enzymes are endowed of impressive turnover frequencies and their main limitation, among the organisms, is the oxygen sensibility. However, [FeFe]-Hydrogenase of Clostridium beijeirinckii (CbA5H) is capable to form an inactive state protected from O2 damage. In this work we investigated with protein film voltammetry (PFV) the properties of CbA5H wildtype and mutants (A419S, I424T-A425S, I424N-A425S, I424N and C236A) , produced in our laboratories, in particular their potential-dependent reactivation kinetics that act as not-chemical reducing species demonstrating that is possible reactivate the enzyme by reductive potential on the electrode surface. We provided some electrochemical method to estimate enzyme bias and the changes reported by mutants compared with wildtype. Differences founded between mutant and wildtype potential-dependent reactivation constants data were also fitted with mono- and biexponential functions to establish which was the best model to describe the dependency. However, electrochemistry provides good information only about activation and inactivation dynamics but not about specific activity or turnover frequency. Indeed, the main limitation is the unknown electroactive coverage. This work provides useful electrochemical profiles of wildtype and specific mutants that necessarily have to be integrated with solution assay studies of the enzymes to completely understand CbA5H behaviour with the objective to obtain artificially improved enzymes adapt for enzymatically hydrogen production or in hydrogen-to-electricity reversible conversion systems.